1. Field of the Invention
This invention relates to processes of flocculating cell material from suspending media. It is often desired to separate cell material (such as cells and/or cell debris) from a liquid suspending medium containing the cell material. One way of doing this is to flocculate the cell material so that the flocs formed can be separated from the liquid suspending medium. After separation the cell material itself may be further used. Alternatively the cell material can be discarded and the contents of the suspending medium can be used.
2. Description of the Related Art
It has, however, proved difficult to find flocculation systems which give adequate flocculation performance in the suspending media from which cell material must often be separated. In particular, it is often necessary to separate cell material from complex media such as growth medium. It has been found that flocculation using standard flocculants such as polymers is problematic in these environments.
Sitkey et al in Biotechnology Techniques, Vol. 6, no. 1, 49–52 (1992) describe the removal of cells, solids and colloids from fermentation broth. The aim of the separation is to recover extracellular enzymes present in the suspending medium. Sixteen polymeric materials are described for use as flocculating agents. The types of flocculant used are weakly cationic, medium cationic, strong cationic, weakly anionic, medium anionic and non-ionic. However, according to the authors only two polymers gave effective clarification. These were medium anionic polymers Sedipur T1 and Sedipur TF5, available from BASF. The addition of various cationic and non-ionic polymers is described. Each is added as a single dose as the only flocculant and is ineffective in providing clarification of the fermentation broth.
Mukhopadhyay et al in Biotechnology Techniques, vol. 4, no. 2, 121–126 (1990) also attempt to separate suspended solids from a fermentation broth in order to retrieve extracellular enzymes dissolved in the suspending medium. The authors use various different systems in order to improve coagulation or flocculation. The flocculating agents used were glacial acetic acid, calcium chloride, aluminium sulphate and cationic polyacrylamide. Systems of these were also used in which two or more of these agents were added to the suspending medium. In particular, the authors describe systems in which aluminium sulphate and cationic polyacrylamide are used as the two flocculating agents. The amounts of the cationic polyacrylamide used in the exemplified systems were 0.1, 0.3 and 0.5 g/l (100, 300 and 500 ppm). The amount of aluminium sulphate used was always 5.0 g/l (5000 ppm). Although clarification of the fermentation broth was obtained using this system, the authors state that under high shear the flocs formed were disintegrated into smaller particles. This prevents settling. Thus this system can clarify the fermentation broth but does not provide robust flocs. For this reason the processes which can subsequently be used for separating the flocs from the supernatant are limited. In particular the authors advise against even moderate centrifugation.
The publication EP-A448,926 discloses a system for the flocculation of enzyme. Cell material such as cells and cell debris are removed from a suspending medium such as fermentation broth by mechanical means such as centrifugation and the enzyme which remains in the supernatant is flocculated using a particular flocculating agent. The flocculating agent is a blend of a Mannich acrylamide polymer and a diallyl dimethyl ammonium halide polymer. In the system described, it is necessary to remove cell material first from the fermentation broth by mechanical means so that it does not contaminate the later chemically precipitated products.
Weir et al in Biotechnology Techniques, Vol 7, No. 3 (March 1993) pp 199–204, disclose flocculation of cells from fermentation broth using chitosan, a cationic polyelectrolyte reported to be neutral above pH 7.9. The same authors also describe, in Biotechnology Techniques, Vol 8, No. 2 (February 1994) pp 129–132, the use of various anionic polymers as a pretreatment before use of chitosan as a flocculent.
It is known that flocculation of microbial cells in a liquid culture medium by cationic polyelectrolytes aids separation of the cells from the medium. When the medium contains high concentrations of anionic polyelectrolytes as constituents of the medium and/or produced by the cells, the addition of high molecular weight cationic flocculants produces flocs. These flocs will be contaminated with a mixture of concentrated flocculent and polyelectrolyte complex formed from an anionic and cationic polymer (polysalt) and/or precipitate of an anionic and cationic polymer that causes the flocs to stick to processing equipment. An alternative procedure of adding low molecular weight cationic polymer flocculants to bacterial cultures containing high concentrations of anionic polyelectrolytes requires high polymer doses if adequate flocculation is to be achieved because of the formation of anionic and cationic polymer complex and/or precipitates. Moreover, flocs produced using low molecular weight cationic polymer flocculants have been found to be considerably weaker than flocs produced with high molecular weight polymers. Under shear the cells are released from the flocs produced using low molecular weight cationic polymer flocculants and this results in a reduction in separation efficiency.
It would be desirable to be able to provide an efficient system for the separation of cell material from suspending medium such as fermentation broth. It would also be desirable to be able to use a variety of separation methods after flocculation and to be able to provide flocs robust enough to withstand the separation methods and, if necessary, subsequent use.